Previous work has led to the hypothesis that elevated extracellular Ca2+ concentration inhibits tetanus tension by causing the inward propagation of excitation along the t-tubules in frog skeletal muscle to become decremental. This hypothesis will be tested by microscopic observation of tetanically contracting muscle fibers in the range of sarcomere length 1.6 - 1.95 um. The appearance of wavy myofibrils in the axial core of the fibers will provide evidence for decremental conduction in the t-system. This phenomenon will be explored further with both microscopic and tension measuring experiments with regard to the effects of other multivalent cations, the significance of sarcomere length and fiber diameter and the frequency of stimulation. The importance of this hypothesis is that it suggests that the weakest link in the chain of events of excitation-contraction coupling may be t-tubular conduction. A second objective is to determine if the hexavalent cation, ruthenium red (RR) does gain access to the t-tubules and shift mechanical threshold as reported in the literature. Previous work leads us to believe that RR does not enter the t-system and does not affect mechanical threshold and that the reported shifts may arise from contaminants of commercial RR preparations. Mechanical threshold will be measured both with potassium contractures and direct observation of fibers during the passage of depolarizing current pulses. We will compare the effects of crude RR, purified RR and certain degradation products of RR. The information is important for the cntinued use of RR as a probe in cellular studies.